Project Summary/Abstract This proposal addresses mechanisms used by the histone chaperones FACT and Spt6 to promote assembly, disassembly, and repair of chromatin. Understanding how chromatin is formed and maintained is central to understanding regulation of transcription, DNA replication, and repair. The yeast Saccharomyces cerevisiae is used as a model system that has powerful genetic, biochemical, and genomic tools available to study fundamental processes common to all eukaryotes. The highly collaborative approach proposed here takes full advantage of these tools by using a broad range of methods simultaneously. FACT uses multiple histone- binding domains to convert nucleosomes into an alternative structural form (the reorganized nucleosome) in which both the DNA and the histones are more accessible. It does this without ATP hydrolysis, distinguishing it from chromatin remodelers. Reorganization is reversible, so FACT can also assemble nucleosomes out of loosely associated DNA and histones to construct chromatin. Spt6 also binds histones, DNA, and nucleosomes, but does not induce reorganization. Comparing the activities and functions of these two chaperones will help us understand how each of these factors can both block histone interactions and promote them at the appropriate times, yet each has unique physiological roles that the other cannot perform. FACT has been viewed as a housekeeping factor that promotes transcription and DNA replication by loosening each nucleosome encountered to allow polymerases to progress through chromatin. This view has been challenged and we are in the process of replacing it with a model in which FACT acts to establish and maintain chromatin in a precisely optimized architecture, and to restore this form after disturbances like transcription or replication. A key goal of this proposal is to continue challenging the existing model and to contribute to proposing and testing the new one. Aim 1 addresses questions about what drives the differential localization of FACT and Spt6, and how they promote or inhibit nucleosome turnover locally to sculpt chromatin structure genome-wide. Reorganization can promote assembly or disassembly of nucleosomes, but we do not know what the reorganized form looks like or what influences how it is resolved. Aim 2 examines this using two distinct single molecule methods in collaborations with the Ha and Studitsky labs, as well as our established and emerging biochemical assays examining the features of nucleosomes, histones, and chaperones that affect reorganization. Aim 3 examines the role of the DNA binding module that is common to all FACT complexes, but whose architecture varies among species. Together these approaches will provide answers to persistent questions about the mechanisms used by histone chaperones, as well as new questions about their physiological roles.